Whether apoptotic stimuli arise from the nucleus, cell membrane surface, or the mitochondria, ultimately, the stimuli converge on a process of activation of a family of cysteine proteases known as the caspases (cysteine aspartases). Activation of caspase family members mediate programmed cell death in normal physiology and a number of diseases with caspase-3 standing at the center of the execution pathway of the cell death program. Existing in a non-active pro-enzyme form in the cytosol of resting cells, caspase-3 is one key "effector" protease when activated. Thus, to monitor the final commitment of tumor cells to death pathways, the need exists to directly quantify the enzymatic activity of caspase-3 in vivo. To meet this challenge, we designed and synthesized a new class of peptide based imaging agents that can permeate across: the cell membrane into the cytosolic compartment. We have now discovered non-native permeation motifs with 10-fold greater membrane permeation properties enabling enhanced imaging signals. Furthermore, redesigned as small molecular weight optical imaging agents containing quenched fluorophores flanking target peptide sequences, the permeation peptides are activated by caspase-3. Upon cleavage, these agents show caspase-3-dependent fluorescence signal amplification, thereby enabling high quality enzyme-specific molecular imaging of intracellular processes in vivo. We propose characterization of each of the three components of our targeted peptide imaging conjugates: the permeation motif, the activatable linker, and the fluorophore/quencher pairs. We propose to use a degenerate peptide combinatorial library to determine preferred recognition sequences of the peptide imaging substrates, define the structural determinants and charge of retained peptides that confer favorable cell retention, and characterize dual-labeled peptides for multi-modality or multi-wavelength analysis. We will perform pre-clinical evaluation of peptide conjugates to study the time course of activation of caspase-dependent apoptosis during therapy in tumor models in vivo. Studies with these novel imaging tools will assist interrogation of the efficacy of new molecular targeted therapies in cancer.